A major roadblock in the development of off-the-shelf, small-caliber vascular grafts is achieving rapid endothelialization of the scaffold while minimizin the risk of thrombosis, intimal hyperplasia, and mechanical failure. Given that platelet aggregation and smooth muscle cell proliferation may be mediated by controlling endothelial cell (EC) growth and phenotype, the development of materials that direct appropriate EC behavior would have a significant impact on small vessel repair and replacement. However, matrix properties which promote graft endothelialization may not be consistent with those appropriate to sustain the loads associated with adult vasculature. To address this limitation, we propose to fabricate multilayered hydrogel-electrospun mesh scaffolds in which a hydrogel layer provides a local environment inductive of rapid endothelialization and an electrospun mesh sleeve provides bulk strength, compliance matching, and suture retention. Thus, each component can be individually tuned to achieve improved outcomes without detriment to other design goals. We propose to circumvent the limitations associated with native biopolymer gels by generating novel bioactive hydrogels using the collagen-mimetic protein Scl2.28 (Scl2). Scl2 is a recently discovered protein which has the triple helical structure characteristic of native collagen but lacks collagen's array of cell adhesion, cytokine binding, and enzyme-cleavage sites. For the present work, we have introduced 11 and 21 adhesion sites into the parent Scl2 to provide a mechanism for EC interactions while maintaining the low platelet aggregation associated with Scl2. Scl2-based hydrogel formulations that induce desired cell behaviors will be utilized in the fabrication of the multilayer vascular graft reinforced with non-degradable electrospun mesh sleeves designed to have mechanical properties similar to native coronary arteries. Aim 1. Identify PEGDA-Scl2 compositions that promote rapid endothelialization of the vascular graft (adhesion, migration, quiescent phenotype) while maintaining the non-thrombogenic nature of Scl2 proteins. Aim 2. Fabricate a multilayer vascular graft with clinically-relevant mechanical properties (burst pressure, suture retention strength, compliance) by reinforcing hydrogels with electrospun polyurethane sleeves. Aim 3. Assess biocompatibility and biostability of each component of the composite graft. Aim 4. Evaluate multilayer grafts in vivo after implantation as carotid grafts in a Yucatan miniature pig model. At the end of the 5 year period, we will have evaluated these new conduits in preclinical animal studies and demonstrated their potential utility as off-the-shelf, small-caliber vascular grafts. From a fundamental perspective, this family of hybrid materials will provide the tools to elucidate endothelialization processes critical to the clinical success of numerous cardiovascular devices. Furthermore, the control over both bioactivity and modulus afforded by PEGDA-Scl2 gels, combined with the ability to target a range of different cell types by incorporating different integrin binding motifs into Scl2, will form a powerful platform in the creation of new bioactive materials for a wide range of biomedical applications. PUBLIC HEALTH RELEVANCE: Cardiovascular diseases, including coronary artery disease and peripheral arterial disease, affect approximately 1 in 3 Americans and remain the leading cause of mortality in the United States. Bioactive vascular grafts have the potential to replace damaged arteries without the complications associated with autologous or current synthetic grafts. At the end of the 5 year period, we will have evaluated bioactive, multilayer grafts in preclinical animal studies and demonstrated the potential utility of this novel design as an off-the-shelf, small-caliber vascular graft. From a fundamental perspective, the development of the proposed family of hybrid materials will provide the tools to elucidate endothelialization processes critical to the clinical success of numerous cardiovascular devices.